1. Field of the Invention
The disclosure below relates to the use of a protein export system. The disclosed system provides effective methods and compositions useful for the production of recombinant proteins.
2. Description of the Related Art
Protein expression systems have long used high copy number expression plasmids or expression vectors in an attempt to increase yields of recombinant proteins of interest. High copy number expression plasmids and the proteins of interest they encode can exert a negative effect on the fitness of a host containing an expression plasmid. The notable burden placed upon prokaryotic host cells carrying multicopy plasmids is the cumulative result of a metabolic cascade triggered by two processes: 1) the replication and maintenance of expression plasmids and 2) transcription and translation of the various plasmid-encoded functions including the gene of interest. Such mechanisms could explain the observation that plasmid-bearing bacteria grow slower than plasmid-less bacteria. This burden can also explain the observation that growth rate decreases as copy number increases.
As the gene of interest is expressed, the growth rate of the recombinant host cell decreases. The decrease in growth rate may trigger the induction of various cellular proteases that can degrade recombinantly produced protein present in cytoplasm of the host cell. Reduced growth rate is therefore the inevitable consequence of metabolic burden, which in turn is the cumulative result of a number of physiological perturbations. Because this reduction in the growth rate creates a selective pressure for loss of resident plasmids in the absence of selection, significant loss of expression plasmids from the host cell carrying an expression vector may occur after transformation of the host cell.
Host cells with reduced growth rates can spontaneously shed an expression plasmid to remove from the host cell an unnecessary metabolic burden and allow plasmid-less host cells to quickly outgrow the population of plasmid-bearing host cells. Such a shift in protein expression within a population of host cells would be expected to reduce the protein production.
Accordingly, it would be desirable to prepare a protein expression system that would optimize protein expression from the expression vector while minimizing the metabolic burden on the host cell generated by the expression vector.